The organization of the chromatin fiber within the nucleus is an important aspect of the expression of eukaryotic genes, but little is known about the mechanisms regulating the establishment and maintenance higher-order chromatin domains. Chromatin insulators or boundary elements appear to be important players in the establishment of these domains, as they are thought to regulate the expression of eukaryotic genes by controlling the organization of the DNA within the nucleus. The gypsy insulator is perhaps the best-studied example of this class of sequences. It is composed of 12 DNA binding sites for the Su(Hw) protein. A second component of the gypsy insulator, the Mod(mdg4)2.2 protein, does not bind to DNA directly and its presence in the insulator is due to its direct interaction with Su(Hw). The protein components the insulator are present at several hundred sites on polytene chromosomes, but in interphase nuclei the proteins concentrate at approximately 25 sites located mostly in the nuclear periphery, causing the chromatin fiber to form rosette-like structures. In this application, we propose to continue investigating the mechanisms of insulator function by studying two new additional protein components of the gypsy insulator named CP190 and CG15104. These proteins will be characterized and the biochemical basis for their interaction with Su(Hw) and Mod(mdg4)2.2 will be studied in detail. The role of these proteins in molecular and cellular aspects of insulator function will be determined. In addition, endogenous insulators, distinct from that present in the gypsy retrotransposon, will be characterized in order to derive an insulator map of the genome. This physical map of insulators will be compared to transcriptional maps of the Drosophila genome. The allocation of all the insulators in the X chromosome to specific rosette structures will be analyzed. This arrangement will be contrasted between cells from different tissues. Experiments to visualize chromatin domains in vitro and in vivo will also be carried out, and the effect of various insulator proteins on their formation will be analyzed. From these studies, we hope to gain insights into the organization of the DNA in the nucleus and its role in the control of gene expression in eukaryotes. [unreadable] [unreadable]